AbstractCellular senescence is a cell fate characterised by the permanent arrest from the cell cycle with an altered, mainly proinflammatory, secretory phenotype. Although, a positive process in development and regeneration, the accumulation of senescent cells, as seen in tissues of aged humans, can cause many negative effects. Within skeletal muscle the resident stem cells, Pax7+ve and CD56+ve satellite cells / muscle precursor cells (MPCs), are essential for muscle maintenance and repair following damage. Impairments either in their inherent behaviour, such as by being senesced, or by being negatively influenced by their environment, such as by the senescence associated secretory phenotype (SASP) of local senescent cells, may impair muscle regeneration in later life and contribute to the age-related loss of skeletal muscle mass, sarcopenia. Furthermore, regeneration is also dependent on the interaction of MPCs with other key cell types in muscle, principally fibroblasts, therefore understanding the senescent characteristics of both cell types is essential to gain further insight into skeletal muscle ageing. Currently, little is known about the senescent phenotype of human skeletal muscle derived cells.
The aim of the work in this thesis was thus to investigate the senescent phenotype of human primary skeletal muscle derived MPCs and fibroblasts in a primary cell culture system. Once characterised this phenotype would then be used to investigate senescence within skeletal muscle cells obtained from very old and frail hip fracture patients. MPCs were sorted using CD56+ve magnetic beads after extraction and expansion from muscle biopsy samples taken from young healthy male volunteers (n=6, 22±1 years). Fibroblasts were derived from the negative fraction. In the first series of experiments cumulative population doublings (CPD) across multiple passages was used to induce cellular replicative senescence (RS). In the second, cells were treated with Doxorubicin (DOX; 0.2 μM) for 24h to induce a senescent phenotype and then investigated at a number of timepoints up to 35 days post treatment. In both approaches, senescence was determined by the presence of senescence-associated β-galactosidase (SA β-gal) and the protein expression of p16, together with the absence of proliferation marker, Ki67. With expression determined using custom built cell-by-cell image analysis programs. The mRNA levels of established SASP factors (e.g. PAI-1, TGF-β1, IL-8 and IGFBP-3, amongst others) were also measured as was the differentiation capacity of MPCs for myogenic fusion.
In the RS study only two of the six MPC populations remained highly myogenic through to RS. Senescence of these two populations of MPCs was shown by increased number of SA β-gal-positive and p16 expressing cells, and decreased Ki67 expression. The senescent, late passage MPCs showed a markedly reduced myogenic fusion index, compared to proliferative MPCs. The fibroblast experiments were terminated at the same time as the equivalent MPC cultures reached senescence. At this point, none of the fibroblast populations had reached RS, as evidenced by no changes in SA β-gal or p16, although there was a trend for slower population doubling times when compared to early passage rates and significantly decreased Ki67. When both MPCs and fibroblasts were exposed to DOX for 24h, senescence was demonstrated shortly after, with 99% of both cell types being SA β-gal-positive four days after DOX treatment, Ki67 expression markedly reduced and MPCs being less able to fuse and form myotubes. SASP factor (i.e. IGFBP7, PAI-1, IGFBP3) mRNA expression from this 4-day time point until 35 days post-DOX treatment varied over time and magnitude in both MPCs and fibroblasts. MPCs isolated from skeletal muscle of old (82±9 yrs) hip fracture patients showed significant differences in senescence marker and SASP factor expression, compared to young donor cells after a similar time in culture.
In conclusion, RS is hard to achieve in human primary muscle cultures because even highly purified MPCs populations can eventually be overrun by initially small populations of fibroblasts, whilst the sorted fibroblast cultures, from the same biopsy samples, appear to take a much longer time to reach RS than the MPCs. DOX treatment allows a synchronised and rapid induction of senescence in both cell types eliminating these problems. Here the results showed both varying temporal and quantitative responses of the different SASP factors between the two cell types. The data highlight the complexity and dynamism of the senescent state within different cell types derived from human skeletal muscle. When myoblasts are taken from a very elderly frail human population undergoing hip fracture surgery, markers of senescence are elevated compared to young myoblasts after similar exposure to cell culture. These findings suggest that cellular senescence may start to be an issue within skeletal muscle cells very late in life. Therefore, this work could contribute to supporting research into healthy human ageing by investigating strategies which prevent the accumulation, or remove, these senescent cells.
|Date of Award
|1 Sept 2020
|Stephen Harridge (Supervisor) & Georgina Ellison (Supervisor)